技术资料

Aldehyde dehydrogenase 1A1 (ALDH1A1) activity is used as a marker of breast cancer stem cells; however,little is known about the regulation of ALDH1A1 expression. Mucin 1 (MUC1) is a heterodimeric protein that is aberrantly overexpressed in most human breast cancers. In studies of breast cancer cells stably silenced for MUC1 or overexpressing the oncogenic MUC1-C subunit,we demonstrate that MUC1-C is sufficient for induction of MEK → ERK signaling and that treatment with a MUC1-C inhibitor suppresses ERK activation. In turn,MUC1-C induces ERK-mediated phosphorylation and activation of the CCAAT/enhancer-binding protein β (C/EBPβ) transcription factor. The results further show that MUC1-C and C/EBPβ form a complex on the ALDH1A1 gene promoter and activate ALDH1A1 gene transcription. MUC1-C-induced up-regulation of ALDH1A1 expression is associated with increases in ALDH activity and is detectable in stem-like cells when expanded as mammospheres. These findings demonstrate that MUC1-C (i) activates a previously unrecognized ERK→C/EBPβ→ALDH1A1 pathway,and (ii) promotes the induction of ALDH activity in breast cancer cells. View Publication

The molecular mechanisms underlying the differentiation of neural progenitor cells (NPCs) remain poorly understood. In this study we investigated the role of Ca(2+) and cAMP (cyclic adenosine monophosphate) in the differentiation of NPCs extracted from the subventricular zone of E14.5 rat embryos. Patch clamp recordings revealed that increasing cAMP-signaling with Forskolin or IBMX (3-isobutyl-1-methylxantine) significantly facilitated neuronal functional maturation. A continuous application of IBMX to the differentiation medium substantially increased the functional expression of voltage-gated Na(+) and K(+) channels,as well as neuronal firing frequency. Furthermore,we observed an increase in the frequency of spontaneous synaptic currents and in the amplitude of evoked glutamatergic and GABAergic synaptic currents. The most prominent acute effect of applying IBMX was an increase in L-type Ca(2+)currents. Conversely,blocking L-type channels strongly inhibited dendritic outgrowth and synapse formation even in the presence of IBMX,indicating that voltage-gated Ca(2+) influx plays a major role in neuronal differentiation. Finally,we found that nifedipine completely blocks IBMX-induced CREB phosphorylation (cAMP-response-element-binding protein),indicating that the activity of this important transcription factor equally depends on both enhanced cAMP and voltage-gated Ca(2+)-signaling. Taken together,these data indicate that the up-regulation of voltage-gated L-type Ca(2+)-channels and early electrical excitability are critical steps in the cAMP-dependent differentiation of SVZ-derived NPCs into functional neurons. To our knowledge,this is the first demonstration of the acute effects of cAMP on voltage-gated Ca(+2)channels in NPC-derived developing neurons. View Publication

BACKGROUND: A few reports suggested that low levels of Wnt signaling might drive cell reprogramming,but these studies could not establish a clear relationship between Wnt signaling and self-renewal networks. There are ongoing debates as to whether and how the Wnt/β-catenin signaling is involved in the control of pluripotency gene networks. Additionally,whether physiological β-catenin signaling generates stem-like cells through interactions with other pathways is as yet unclear. The nasopharyngeal carcinoma HONE1 cells have low expression of β-catenin and wild-type expression of p53,which provided a possibility to study regulatory mechanism of stemness networks induced by physiological levels of Wnt signaling in these cells.backslashnbackslashnRESULTS: Introduction of increased β-catenin signaling,haploid expression of β-catenin under control by its natural regulators in transferred chromosome 3,resulted in activation of Wnt/β-catenin networks and dedifferentiation in HONE1 hybrid cell lines,but not in esophageal carcinoma SLMT1 hybrid cells that had high levels of endogenous β-catenin expression. HONE1 hybrid cells displayed stem cell-like properties,including enhancement of CD24(+) and CD44(+) populations and generation of spheres that were not observed in parental HONE1 cells. Signaling cascades were detected in HONE1 hybrid cells,including activation of p53- and RB1-mediated tumor suppressor pathways,up-regulation of Nanog-,Oct4-,Sox2-,and Klf4-mediated pluripotency networks,and altered E-cadherin expression in both in vitro and in vivo assays. qPCR array analyses further revealed interactions of physiological Wnt/β-catenin signaling with other pathways such as epithelial-mesenchymal transition,TGF-β,Activin,BMPR,FGFR2,and LIFR- and IL6ST-mediated cell self-renewal networks. Using β-catenin shRNA inhibitory assays,a dominant role for β-catenin in these cellular network activities was observed. The expression of cell surface markers such as CD9,CD24,CD44,CD90,and CD133 in generated spheres was progressively up-regulated compared to HONE1 hybrid cells. Thirty-four up-regulated components of the Wnt pathway were identified in these spheres.backslashnbackslashnCONCLUSIONS: Wnt/β-catenin signaling regulates self-renewal networks and plays a central role in the control of pluripotency genes,tumor suppressive pathways and expression of cancer stem cell markers. This current study provides a novel platform to investigate the interaction of physiological Wnt/β-catenin signaling with stemness transition networks. View Publication

The p53 tumor suppressor protein is a major sensor of cellular stresses,and upon stabilization,activates or represses many genes that control cell fate decisions. While the mechanism of p53-mediated transactivation is well established,several mechanisms have been proposed for p53-mediated repression. Here,we demonstrate that the cyclin-dependent kinase inhibitor p21 is both necessary and sufficient for the downregulation of known p53-repression targets,including survivin,CDC25C,and CDC25B in response to p53 induction. These same targets are similarly repressed in response to p16 overexpression,implicating the involvement of the shared downstream retinoblastoma (RB)-E2F pathway. We further show that in response to either p53 or p21 induction,E2F4 complexes are specifically recruited onto the promoters of these p53-repression targets. Moreover,abrogation of E2F4 recruitment via the inactivation of RB pocket proteins,but not by RB loss of function alone,prevents the repression of these genes. Finally,our results indicate that E2F4 promoter occupancy is globally associated with p53-repression targets,but not with p53 activation targets,implicating E2F4 complexes as effectors of p21-dependent p53-mediated repression. View Publication

The optical isomers of the 1,4-dihydropyridine BAY K 8644 were studied in isolated rabbit aorta and heart preparations. The (-)-enantiomer has the known vasoconstricting and positive inotropic properties of the Ca agonistic compound. In contrast,its antipode shows at about 10-50 times higher concentrations the vasodilating and negative inotropic effects of Ca antagonistic drugs. It is concluded that neither simple chemical nor physical actions can be responsible for the opposite effects of Ca antagonistic and Ca agonistic dihydropyridines. View Publication

Biochemical factors can help reprogram somatic cells into pluripotent stem cells,yet the role of biophysical factors during reprogramming is unknown. Here,we show that biophysical cues,in the form of parallel microgrooves on the surface of cell-adhesive substrates,can replace the effects of small-molecule epigenetic modifiers and significantly improve reprogramming efficiency. The mechanism relies on the mechanomodulation of the cells' epigenetic state. Specifically,decreased histone deacetylase activity and upregulation of the expression of WD repeat domain 5 (WDR5)—a subunit of H3 methyltranferase—by microgrooved surfaces lead to increased histone H3 acetylation and methylation. We also show that microtopography promotes a mesenchymal-to-epithelial transition in adult fibroblasts. Nanofibrous scaffolds with aligned fibre orientation produce effects similar to those produced by microgrooves,suggesting that changes in cell morphology may be responsible for modulation of the epigenetic state. These findings have important implications in cell biology and in the optimization of biomaterials for cell-engineering applications. View Publication

PURPOSE Cancer stem cells (CSC) are the tumorigenic cell population that has been shown to sustain tumor growth and to resist conventional therapies. The purpose of this study was to evaluate the potential of histone deacetylase inhibitors (HDACi) as anti-CSC therapies. EXPERIMENTAL DESIGN We evaluated the effect of the HDACi compound abexinostat on CSCs from 16 breast cancer cell lines (BCL) using ALDEFLUOR assay and tumorsphere formation. We performed gene expression profiling to identify biomarkers predicting drug response to abexinostat. Then,we used patient-derived xenograft (PDX) to confirm,in vivo,abexinostat treatment effect on breast CSCs according to the identified biomarkers. RESULTS We identified two drug-response profiles to abexinostat in BCLs. Abexinostat induced CSC differentiation in low-dose sensitive BCLs,whereas it did not have any effect on the CSC population from high-dose sensitive BCLs. Using gene expression profiling,we identified the long noncoding RNA Xist (X-inactive specific transcript) as a biomarker predicting BCL response to HDACi. We validated that low Xist expression predicts drug response in PDXs associated with a significant reduction of the breast CSC population. CONCLUSIONS Our study opens promising perspectives for the use of HDACi as a differentiation therapy targeting the breast CSCs and identified a biomarker to select patients with breast cancer susceptible to responding to this treatment. View Publication

We present an integrated platform comprised of a biomimetic substrate and physiologically aligned human pluripotent stem cell-derived cardiomyocytes (CMs) with optical detection and algorithms to monitor subtle changes in cardiac properties under various conditions. In the native heart,anisotropic tissue structures facilitate important concerted mechanical contraction and electrical propagation. To recapitulate the architecture necessary for a physiologically accurate heart response,we have developed a simple way to create large areas of aligned CMs with improved functional properties using shrink-wrap film. Combined with simple bright field imaging,obviating the need for fluorescent labels or beads,we quantify and analyze key cardiac contractile parameters. To evaluate the performance capabilities of this platform,the effects of two drugs,E-4031 and isoprenaline,were examined. Cardiac cells supplemented with E-4031 exhibited an increase in contractile duration exclusively due to prolonged relaxation peak. Notably,cells aligned on the biomimetic platform responded detectably down to a dosage of 3nm E-4031,which is lower than the IC50 in the hERG channel assay. Cells supplemented with isoprenaline exhibited increased contractile frequency and acceleration. Interestingly,cells grown on the biomimetic substrate were more responsive to isoprenaline than those grown on the two control surfaces,suggesting topography may help induce more mature ion channel development. This simple and low-cost platform could thus be a powerful tool for longitudinal assays as well as an effective tool for drug screening and basic cardiac research. ?? 2013 Elsevier Ltd. View Publication

BACKGROUND Cancer development results from the progressive accumulation of genomic abnormalities that culminate in the neoplastic phenotype. Cytogenetic alterations,mutations and rearrangements may be considered as molecular legacy which trace the clonal history of the disease. Concomitant tumors are reported and they may derive from a common or divergent founder clone. B-cell chronic lymphocytic leukemia (B-CLL) and plasma cell myeloma (PCM) are both mature B-cell neoplasms,and their concomitancy,albeit rare,is documented. CASE PRESENTATION Here,we described a patient with prior B-CLL with secondary development of PCM. Cytogenetic and multi parametric flow cytometry analyses were performed. The B-CLL population presented chromosome 12 trisomy,unlikely the arisen PCM population. CONCLUSION The close follow up of B-CLL patients is important for early intervention in case of development of other malignancy,such as myeloma. Our observation suggests these two diseases may have arisen from different clones. We understand that the investigation of clonal origin may provide important information regarding therapeutic decisions,and should be considered in concomitant neoplasm. View Publication

Stem cell differentiation is regulated by complex repertoires of signaling ligands which often use multivalent interactions,where multiple ligands tethered to one entity interact with multiple cellular receptors to yield oligomeric complexes. One such ligand is Sonic hedgehog (Shh),whose posttranslational lipid modifications and assembly into multimers enhance its biological potency,potentially through receptor clustering. Investigations of Shh typically utilize recombinant,monomeric protein,and thus the impact of multivalency on ligand potency is unexplored. Among its many activities,Shh is required for ventralization of the midbrain and forebrain and is therefore critical for the development of midbrain dopaminergic (mDA) and forebrain gamma-aminobutyric acid (GABA) inhibitory neurons. We have designed multivalent biomaterials presenting Shh in defined spatial arrangements and investigated the role of Shh valency in ventral specification of human embryonic stem cells (hESCs) into these therapeutically relevant cell types. Multivalent Shh conjugates with optimal valencies,compared to the monomeric Shh,increased the percentages of neurons belonging to mDA or forebrain GABAergic fates from 33% to 60% or 52% to 86%,respectively. Thus,multivalent Shh bioconjugates can enhance neuronal lineage commitment of pluripotent stem cells and thereby facilitate efficient derivation of neurons that could be used to treat Parkinson's and epilepsy patients. View Publication

A significant clinical need exists to differentiate human pluripotent stem cells (hPSCs) into cardiomyocytes,enabling tissue modeling for in vitro discovery of new drugs or cell-based therapies for heart repair in vivo. Chemical and mechanical microenvironmental factors are known to impact the efficiency of stem cell differentiation,but cardiac differentiation protocols in hPSCs are typically performed on rigid tissue culture polystyrene (TCPS) surfaces,which do not present a physiological mechanical setting. To investigate the temporal effects of mechanics on cardiac differentiation,we cultured human embryonic stem cells (hESCs) and their derivatives on polyacrylamide hydrogel substrates with a physiologically relevant range of stiffnesses. In directed differentiation and embryoid body culture systems,differentiation of hESCs to cardiac troponin T-expressing (cTnT+) cardiomyocytes peaked on hydrogels of intermediate stiffness. Brachyury expression also peaked on intermediate stiffness hydrogels at day 1 of directed differentiation,suggesting that stiffness impacted the initial differentiation trajectory of hESCs to mesendoderm. To investigate the impact of substrate mechanics during cardiac specification of mesodermal progenitors,we initiated directed cardiomyocyte differentiation on TCPS and transferred cells to hydrogels at the Nkx2.5/Isl1+ cardiac progenitor cell stage. No differences in cardiomyocyte purity with stiffness were observed on day 15. These experiments indicate that differentiation of hESCs is sensitive to substrate mechanics at early stages of mesodermal induction,and proper application of substrate mechanics can increase the propensity of hESCs to differentiate to cardiomyocytes. textcopyright 2013 Acta Materialia Inc. View Publication

Ex vivo expansion of satellite cells and directed differentiation of pluripotent cells to mature skeletal muscle have proved difficult challenges for regenerative biology. Using a zebrafish embryo culture system with reporters of early and late skeletal muscle differentiation,we examined the influence of 2,400 chemicals on myogenesis and identified six that expanded muscle progenitors,including three GSK3$\$,two calpain inhibitors,and one adenylyl cyclase activator,forskolin. Forskolin also enhanced proliferation of mouse satellite cells in culture and maintained their ability to engraft muscle in vivo. A combination of bFGF,forskolin,and the GSK3$\$ BIO induced skeletal muscle differentiation in human induced pluripotent stem cells (iPSCs) and produced engraftable myogenic progenitors that contributed to muscle repair in vivo. In summary,these studies reveal functionally conserved pathways regulating myogenesis across species and identify chemical compounds that expand mouse satellite cells and differentiate human iPSCs into engraftable muscle. View Publication